The goal of the proposed studies is to understand the fundamental mechanisms by which single-subunit RNA polymerases (RNAP) catalyze transcription. Single-subunit RNAPs transcribe the genomes of mitochondria that are found in eucaryotes and parasites and mitochondrial RNAPs are remarkably homologous to the RNAPs encoded by T7/T3 like phages. Understanding the mechanisms of single-subunit RNAPs has applications in developing therapeutics for parasitic infectious diseases and diseases related to mitochondrial defects in humans. We propose to investigate the structure and function of single-subunit RNAPs, from bacteriophage T7 and yeast mitochondrial RNAP using approaches such as transient state kinetics and fluorescence resonance energy transfer (FRET). The essential mechanistic features of transcription catalyzed by single subunit or multisubunit RNAPs are conserved. By choosing enzymatically tractable systems, we are able to study the complex mechanisms of transcription in great detail that will eventually lead to a predictive and quantitative model of transcription. It is recognized that transcription initiation, elongation, and termination all play essential roles in regulating gene expression, both in procaryotes and eucaryotes. Dissecting initiation, promoter clearance, and elongation by determining the rate constants and the energetics of the steps provides the essential framework to understand transcription and its regulation. The proposed studies will be carried out with the following specific aims: 1) To characterize the initial steps of transcription initiation in T7 RNAP. We will use a combination of single molecule FRET, stopped- flow FRET, and a rapid protein mapping method to characterize the structures of intermediates and the dynamics of the transition from initiation to elongation. 2) To investigate the mechanism of transcription by S. cerevisiae mitochondrial RNAP. Using established biophysical approaches from our studies of T7 RNAP, we will investigate the initiation mechanisms of mitochondrial RNAP with the goal of understanding the role of its specificity factor. 3) To characterize the kinetic pathway of transcription elongation. Radiometric quenched-flow, fluorescence stopped-flow, and computational kinetic modeling methods will be used to define the elementary steps of nucleotide addition and to understand the mechanisms of fidelity and strand displacement RNA synthesis during elongation.